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Introduction
Osteosarcoma is the most common primary malignant
bone tumor in children and adolescents. It accounts for 20%
of the malignant tumors of children[1]. During the past 3
decades, the 5-year survival rate has improved significantly
to 60%_70%[2] as a result of the extensive application of
multi-agent chemotherapy. However, more than 30% of patients
still suffer medical failure, mostly because of relapse and
metastases[3]. Skip metastasis is defined as a second focus
of osteosarcoma, anatomically separated from the primary
tumor, which is found in the same bone or on the opposite
side of the adjacent joint[4]. Patients with skip metastasis
suffer more from medical failure, the prognosis of which is
parallel or even worse than those with lung
metastasis[5_7]. Until now, little has been known about the intramedullary
dissemination of osteosarcoma.
Cell lines and clinically-relevant animal models provide
important resources for investigating tumor molecular
pathology and evaluating new therapy
strategies[8_12]. The established osteosarcoma cell lines, especially those with
tumorigenic and metastatic potential, play a considerable role in
the discovery and function analysis of the molecular events
of tumor development and
metastasis[12,13_16]. Up until now, many osteosarcoma cell lines with different background have
been developed, but most of them are not tumorigenic, and
even fewer can metastasize[11]. To our knowledge, no cell
lines originating from skip metastasis have been reported.
Two novel human osteosarcoma cell lines, Zos and
Zos-M, were established respectively from the primary tumor and
the skip lesion of an 18-year-old male patient. The purpose
of the current study is to investigate the characteristics of
these 2 cell lines, including drug sensitivity, so as to provide
useful tools for the study of osteosarcoma. Biological
behaviors and the expression of a small panel of
metastasis-related genes were also compared between the 2 cell lines to
find out potential molecular events that may contribute to
the dissemination of osteosarcoma.
Material and methods
Animals and drugs Male BALB/c nude mice (4_6 weeks)
were obtained from the Experimental Animal Department of
Sun Yat-Sen University (Guangzhou, China) and raised in a
sterile condition with constant temperature and humidity.
All animal experimental protocols were approved by the
Animal Care and Use Committee of Sun Yat-Sen University.
Methotrexate (MTX) and
3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) were purchased from
Sigma (St Louis, MO, USA). Doxorubicin (ADM) and cisplatin (DDP) were purchased from the National Institute
for the Control of Pharmaceutical and Biological Products
(Beijing, China).
Cell source and culture An 18-year-old boy suffered
continuous pain in his right knee for 3 months. A needle
biopsy confirmed the diagnosis of osteoblastic
osteosarcoma in January 2005. After 2 cycles of chemotherapy using
the protocols ADM, DDP, MTX, isofosfamide, alternatively,
the boy rejected the operation. During the following 4
months, the tumor aggravated quickly and reached 40 cm×30
cm×10 cm in size. Hip disarticulation was performed in
August 2005, and lung metastasis were detected by X-ray
photography 1 month later. Three months later, the patient died
of tumor dissemination. Two osteosarcoma specimens were
obtained, respectively, from the primary tumor in the right
distal femoral extremity and skip metastasis in the right femoral
head. Informed consent for the experimental use of the
specimens was obtained from the patient and his parents.
Parts of the specimens were fixed for pathological
examinations and other parts were rinsed several times with
phosphate-buffered saline (PBS) containing penicillin G (100
U/mL) and streptomycin (100 mg/mL). The tumor fragments
were then minced into small pieces, approximately 1
mm3 in size, and seeded into screw-top culture flasks (Corning
Costar, Corning, NY, USA) moistened with medium. The
culture flasks were inverted and cultured at 37 °C in a 5%
CO2 incubator for 2 h, allowing adherence of the explants to
the bottom. They were then turned over and a small amount
of Dulbecco's modified Eagle's medium (DMEM; high glucose,
GIBCO, Invitrogen, Carlsbad, CA, USA) containing 10% fetal
bovine serum (FBS; GIBCO, InvitrogenCarlsbad, CA, USA ),
penicillin G (100 U/mL), streptomycin (100 mg/mL), and
L-glutamine (2 mmol/L) were added. Two days later, a few
polygonal cells outgrew the explants. Over the next few
days, the long-spindle-shaped fibroblasts outgrowing
beside the tumor cells were labeled and scraped off. Twelve
days later, the cells reached confluence and were dispersed
by PBS containing 0.25% trypsin and 0.02% EDTA. The cell
suspensions were transferred to new culture flasks to form
monolayer cultures. The cells were maintained in the same
medium and passaged every 3 d.
The human osteosarcoma cell line U-2OS was a gift from
Dr M SERRA (Istituti Ortopedici Rizzoli, Bologna, Italy). It
was cultured in DMEM supplemented with 10% FBS at 37 °C
in a 5% CO2 incubator. The normal osteoblastic cell line
hFOB1.19 (expressing SV40 large T antigen) used for
comparison was originally obtained from the American Type
Culture Collection (Manassas, VA, USA). It was cultured in a
1:1 mixture of Ham's F12 medium and DMEM without phenol
red and with 2.5 mmol/L L-glutamine and 0.3 mg/mL G418.
Morphological observations The morphology of the
living cells in the culture flasks were observed under an
inverted microscope (Olympus CKX31, Olympus, Tokyo,
Japan) and pictures were taken.
The cells for the transmission electron microscope
examination were prepared as described
previously[10]. Briefly, the cells were first fixed in 2.5% glutaraldehyde in 67 mmol/L
phosphate buffer (pH 7.4) and then in 1%
OsO4 in 67 mmol/L phosphate buffer. Both steps were done at 4 °C for 30 min.
Next, the specimens were dehydrated in a graded series of
ethanol concentrations and embedded in Epon 812.
Ultra-thin sections were cut and mounted on nickel mesh grids.
The sections were stained with uranyl acetate and lead
citrate and were examined with a transmission electron
microscope (Hitachi H-800, Tokyo, Japan).
Proliferation and chemosensitivity assay
The cells in the logarithmic growth phase were harvested and seeded
into 6-well plates (Corning Costar, USA) with 5000 cells per
well. Every day for 7 d, the cells in 3 wells were harvested
and counted with trypan blue. The experiment was repeated
3 times. Growth curves were then drawn and the doubling
time was calculated according to the logarithmic phase of
the growth curve.
The sensitivity of MTX was expressed as the ratio of the
concentration resulting in 50% inhibition of growth
(IC50) of the cell lines. To determine the
IC50 values, 5000 cells per well were seeded in triplicate on a 96-well plate with DMEM
containing 10% FBS. The medium was also added to the
wells without cells as the blank control. After 24 h, the
medium in each well was replaced with 180 μL DMEM
containing 10% FBS and no MTX (control) or with different
concentrations of MTX. The MTT assay was done by adding 20
μL MTT(5 mg/mL) into each well 96 h later. After incubation at
37 °C for an additional 4 h, the medium was replaced with 150
μL DMSO to dissolve the formazan that had precipitated. Then
the absorbance was measured at 490 nm in a microtiter plate
reader (elx800 BIO-TEK, Winooski, VT, USA). The
percentages of growth inhibition compared with the appropriate
control were estimated and the IC50 values were calculated from
the cytotoxicity curves (Bliss's software,Bliss Co, CA, USA).
The sensitivity of the other drugs was evaluated as described
for MTX. Data shown is representative of 3 independent
experiments; values presented are the mean±SD. Statistical
significance was determined by Student's t-test using
Microsoft Excel software (Microsoft, Redmond,WA USA). A
2-side P-value of <0.01 was considered statistically significant.
Matrigel invasion assay The transwell chamber with
polycarbonate filters (8 um pore size; Costar, Cambridge,
MA, USA) coated with the artificial basement membrane
matrigel (12.5 μg per filter; Becton Dickinson Labware,
Waltham, MA, USA) were used to detect the invasive ability
of the Zos and Zos-M cell lines. In total,
1×105 cells in 0.1 mL serum-free medium were seeded in the upper chamber,
and 0.6 mL medium with 15% FBS was added to the lower
chamber. After incubation at 37 °C in 5%
CO2 for 24 h, non-invading cells in the upper chamber were wiped completely
clean with a cotton swab, then the filters were fixed with 10%
formalin for 10 min and washed with PBS. The cells were
stained with hematoxylin and rinsed with water. The number
of cells migrating on the lower surface of the filters was
counted under a microscope in 5 random high-power fields
per membrane. Each assay was performed in triplicate.
Tumorigenicity and experimental metastasis assay
The cells at the sixteenth passage in the exponential phase were
harvested, washed with serum-free medium, and counted.
The cells (5×106) were suspended in 0.2 mL PBS and were
injected subcutaneously near the scapula of 6 nude mice for
each cell line. Each week, the tumor size was measured with
a sliding caliper. Two months later, the mice were killed and
the tumors were fixed with 10% formaldehyde and
embedded in wax. The sections stained with hematoxylin-eosin
were used for pathological examination. The lung and liver
were treated similarly for the detection of metastases.
Both of the in vitro passaged osteosarcoma cell cultures
were harvested and prepared for injection as described above.
The cells were brought to a final concentration of
1×107cells/mL in PBS. An aliquot (200
μL) of cell suspensions was injected into the tail vein of each nude mouse, and 8 nude mice were
used for each cell line. Eight weeks later, the mice were killed
and the lung and liver were fixed for the detection of metastases.
Chromosomal analysis The passaged cells in the
exponential phase of growth were incubated with colchicine
(Sigma, Saint Quentin Fallavier, France) at a final
concentration of 0.8 μg/mL for 4 h and harvested. The cells were then
treated with a hypotonic solution of potassium chloride
(0.075 mol/L) and incubated at 37 °C for 30 min. The cells
were then fixed in acetic acid, methanol (1:3,
v:v), mounted on grease-free, cooled slides, and air dried. Giemsa trysin
banding was performed for the chromosome examination.
RNA extraction and RT-PCR Total RNA was extracted
from cells using TRIZOL (Invitrogen, Carlsbad, CA, USA)
according to the product instructions. The extracted RNA
was quantitated by spectrophotometry and examined using
1.2% agarose gel (Biowest, Spain) electrophoresis.
First-strand cDNA was synthesized with 1 μg of total RNA and a
first-strand cDNA synthesis kit (Toyobo, Osaka, Japan)
according to the manufacturer's protocol. The genes tested
included osteogenic-related genes osteocalcin, osteopontin,
alkaline phosphatase (ALP), and tumor invasiveness-related
genes vil2, cadherin-11, Fas, CD44 neurofibromin 2 (NF2),
and breast cancer metastasis suppressor 1 (BRMS1). The
primer sequences and product sizes are listed in Table 1.
Amplification was performed for 25 cycles to avoid
saturation in a 25 μL reaction volume. For β-actin, ALP, osteopontin,
ezrin, cadherin-11, and CD44 each cycle included 30 s at 94 °C,
30 s at 56 °C, and 30 s at 72 °C. For BRMS1 and Fas, it
included 30 s at 94 °C, 30 s at 60 °C, and 30 s at 72 °C. For
osteocalcin, it included 30 s at 94 °C, 30 s at 62 °C, and 30 s at
72 °C. For NF2, it included 30 s at 94 °C, 30 s at 63 °C, and 30
s at 72 °C. The amplification products were electrophoresed
in a 2% agarose gel containing 0.3 μg/mL ethidium bromide
and were observed under UV light.
Results
Morphology by light microscopy and electron
microscopy The cells showed a heterogeneous appearance under
light microscopy (Figure 1), from spindle to polymorphic,
with large nuclei and 2_3 prominent nucleoli. The
karyokinesis phase was frequent, and cells with double nuclei and
multiple nuclei were seen. Many particles were dispersed in
the cytoplasm. The cells grew in a compact pile and
exhibited loss of anchorage dependence. Compared to Zos,
Zos-M exhibited more filopodial protrusions and membrane ruffles
on the cell membrane.
An examination using the transmission electron
microscopic revealed polygonal cells with atypical large nuclei
and inverted nuclear-cytoplasmic ratios; 2_3 obvious nucleoli
were present in a round nucleus. Abundant organelles,
including mitochondria and dilated rough endoplasmic
reticulum, were seen in the cytoplasm (Figure 1).
Cell proliferation rate and sensitivity to
chemotherapeutic drugs The growth curves for the Zos and Zos-M cell
lines were drawn for each cell line (Figure 2A). Both cell
lines reached a growth plateau on the sixth day of culture;
the saturation density of Zos-M was larger than that for
Zos. The population doubling time measured during the
logarithmic phase was 33.65 h for Zos and 31.58 h for Zos-M.
In order to investigate the sensitivity of Zos and Zos-M
to the current chemotherapy drugs compared to U-2OS, the
MTT assay were performed and the IC50 value of each drug
was calculated (Table 2). There were no statistical difference
between the chemosensitivity of Zos and Zos-M for all of
the drugs tested (P=0.417 for MTX; P=0.289
for ADM; P=0.106 for DDP), although they were more resistant to these drugs than
U-2OS, the IC50 of which was significant lower
(P<0.01).
Tumorigenicity and in vitro and in
vivo invasive potential The tumorigenic abilities of Zos and Zos-M were tested
by subcutaneous inoculation on the backs of the nude mice.
Two months later, tumors formed in the subcutis of all 6 mice
for both cell lines. The tumors derived from Zos-M cells
were relatively hard, and some osteoid tissues could be seen
in the section (Figure 3B); the average size of the tumor
reached approximately 2.35 cm3. The tumors derived from
Zos expanded quickly, and hemorrhage and necrosis soon
occurred; the average volume of the tumor reached 6.77
cm3 within 2 months. Like the original tumor, a rare osteoid could
be observed in the histological section (Figure 3A). No
evidence of metastases to the major organs was found 2 months
after xenotransplantation.
The in vitro invasive potential of Zos and Zos-M was
compared by the matrigel invasion assay. Fetal bovine
serum was used as a chemoattractant in the bottom chamber.
There was nearly a 2.5-fold increase in the number of
invaded cell of Zos-M compared to that of Zos (Figure 2B). In
the experimental metastasis assay, no metastases were found
in mice injected with Zos, but 37.5% (3/8) mice injected with
Zos-M developed distant metastases, 2 mice had solitary
tumors forming superficially on the lung (Figure 3C), and 1
had bone metastasis in the spine, but no dispersed metastases
were found.
Chromosomal analysis Karyotypes of both cell lines
were analyzed by Giemsa trypsin banding. Fifteen metaphase
cells (excluding polyploid ones) were analyzed for each cell
line. The number of chromosomes of the Zos cells ranged
from 55 to 60 (mode number was 57). The number of
chromosomes of Zos-M cells ranged from 50 to 56 (mode number was
52). The karyotype was very complex and various numerical
and structural abnormalities were recognized (Figure 4). Most
involved chromosomal abnormalities, including 1p, 1q, 4p, 5p,
11q, 16p, 17p, and 19q. Both cell lines showed alterations of
del(1) (pter→q11:); t(4;5)(q28; p13); t(3;9)(q12; p12), and there
were also some unidentified marker chromosomes.
Expression of osteoblastic markers and
metastasis-related genes Osteoblast biomarkers were detected in both
cell lines; ALP and osteopontin were highly represented,
and osteocalcin was relatively weak, but definitely expressed
(Figure 5). We compared osteoblasts from the Zos and
Zos-M cell lines for the mRNA coding for ezrin, cadherin-11,
CD44, Fas, NF2, and BRMS1. Zos-M expressed lower levels
of cadherin-11 when compared to Zos (Figure 6). The
expression of CD44was detected in hFOB1.19, but not in
Zos-M or Zos. Fas could barely be detected in Zos and Zos-M
cells, although it was weakly expressed in hFOB1.19. The
expression level of ezrin was similar in Zos, Zos-M, and
osteoblasts; no differences in the expression of NF2 and
BRMS1 were found.
Discussion
In the current study, we describe 2 syngeneic cell lines,
Zos and Zos-M. These two cell lines display the molecular
characteristics of an osteoblast phenotype. The
proliferation assay implies that the cell is in the proliferation-active
state and the cells remain stable in culture for 100 passages
without interruption. Both Zos and Zos-M are tumorigenic
after subcutaneous xenotransplantation in athymic mice. The
Zos-M cell line can also develop distant metastases in the
lung and bone after intravenous injection. The
subcutaneous and lung metastasis masses were similar to the primary
tumor in the morphological and immunohistological examinations. All of these indicate that Zos and Zos-M are
well-established osteosarcoma cell lines.
The chromosome analyses were consistent with a
feature of this tumor that was previously
reviewed[17], namely, complex chromosome abnormalities, including a high
percentage of unidentified marker chromosomes, and
pronounced cell-to-cell variation. However, some common
chromosome changes could be found between the 2 cell lines,
including some commonly involved structural abnormalities
described before[17], such as 1p11~p13.1q11~q12 and 17p.
Until now, no specific diagnostic cytogenetic biomarker has
been identified for osteosarcoma, which has led to the
retardation in developing molecular diagnoses and gene
therapies for osteosarcoma.
The introduction of neoadjuvent chemotherapy
dramatically improves the clinical outcome of patients with
osteosarcoma. It could benefit the non-metastatic patient at
diagnosis, and also could change relapse patterns by
delaying the relapse-free interval and reducing the number of
metastasis nodals in the lung[18,19]. However, for those who
relapse with metastases, it is rarely cured. The 5-year
survival decreases sharply to 23%~28% for those relapsed
patients[20,21]. Presumably, these tumors are drug-resistant.
Moreover, a study suggested that those who relapse after
adjuvant chemotherapy have significantly shorter time for
the further progression of disease and shorter survival time
compared with those who relapsed after surgery
alone[18]. Also, the ratio of extrapulmonary metastases increased after
the introduction of adjuvant
chemotherapy[21], which has a poorer prognosis compared with lung
metastasis[22]. This means that the osteosarcoma cell that survives the
chemotherapy becomes less sensitive to the current protocol and
more aggressive than before. In the current study, we found
in this patient that the tumor specimen obtained in the
surgery had little osteoid tissue and was less differentiated than
that of the core needle biopsy. It can be surmised that these
changes developed to adapt to the selection pressure of the
neoadjuvant chemotherapy. The fact that Zos and Zos-M
are more resistant to the drugs than U-2OS, which is
established prior to the chemotherapy era, also confirmed the
hypothesis. So these cell lines could be interesting models
to screen new drugs to surmount chemoresistance.
Skip metastasis is defined as 2 or more discontinuous
lesions in the same bone; patients of skip metastases and
without other detectable distant metastases are classified as
having stage III disease in the recently revised American
Joint Committee on Cancer( AJCC )staging
system[23]. The incidence of skip metastasis ranges from 1.4% to 25% in
different publications. Dismals outlook were recorded in all
reports[2,4_7,24]. In a large retrospective investigation, the
5-year survival of patients with skip metastasis is approximately
53% when the complete surgery resection was achieved and
neoadjuvant chemotherapy were performed, which is similar
to that of patients with distant metastasis in the same
investigation[7]. In some other studies, even poorer outlooks were
reported. The survival for patients with skip metastasis is
significantly less than the survival for patients with
pulmonary metastases at
presentation[5,6,24]. In the current study,
we compared the biological behavior of cells from primary
and skip lesions. In the case of morphological appearance,
Zos-M has more filopodial protrusions and membrane ruffles
than Zos, which suggest high motility. The proliferation of
Zos-M is more active than that of Zos. Furthermore, Zos-M
is more prone to developing lung and bone metastases than
Zos after intravenous injection. All of these indicate that the
skip metastasis clones may represent the more aggressive
one. The chemosensitivity of Zos-M is similar to that of
Zos, which is consistent with prior results that identical
histological responses to preoperative chemotherapy were
observed in primaries and skips[7].
To investigate the different capacity of invasiveness
between Zos and Zos-M, the expression of a small panel of
metastasis-related genes were analyzed by RT-PCR. The
results indicate that the suppression of cadherin-11 in Zos-M
may be involved in the process of dissemination.
Cadherin-11 is an adhesion molecule, which mediates homophilic
cell_cell adhesion and is significantly expressed in osteoblast
cells[25]. The suppression of cadherin-11 in Zos-M may
decrease the attachment between the osteosarcoma cells, thus
initiating the metastasis process, just as E-cadherin does in
the development of carcinoma[26]. The same result was also
observed by Takeshi [25]with immunohistochemistry: a strong
expression of cadherin-11 in normal osteoblasts, but a faint
expression in osteosarcoma. They continued to prove
using the mouse osteosarcoma cell line that transfection of the
cadherin-11 gene into the high metastasis capacity cell line
LM8 leads to the reduction of lung metastasis in
vivo[13,25]. Therefore, it can be speculated that the decreased
expression of cadherin-11 in Zos-M may confer a greater ability of
the tumor cell to metastasize.
The results also imply that CD44 are depressed in the
Zos and Zos-M cell lines, which is consistent with
discoveries in colon cancer cells that the standard form of CD44 acts
as a tumor suppressor and inhibits the potential for
metastasis[27,28], and in invasive breast cancer patients where CD44
expression may be a favorable prognostic
factor[29]. Fas, in the presence of FasL, could start the process of
apoptosis[30]. The poorly-expressed Fas in the Zos-M and Zos cell lines will evade
apoptosis in the lung, where FasL is abundantly expressed.
In conclusion, Zos-M and Zos are well-established
syngeneic osteosarcoma cell lines with different capacities of
invasiveness. Comparisons of gene expression profiles of
these 2 cell lines will shed light on the study of
intramedullary dissemination of osteosarcoma, and may also provide
insight to the investigation of distant metastases. Allowing
for the nature of insensitivity to chemotherapy, these 2 cell
lines and related animal models would be interesting tools to
screen treatment strategies for aggressive osteosarcoma.
Acknowledgements
We thank Dr M SERRA (Rizzoli institute, Bologna, Italy)
for kindly providing the osteosarcoma cell line U-2OS, and
Quan-sheng ZHU (MD Anderson Cancer Center, Houston,
TX, USA) for linguistic support.
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